JP2011010583A - Liquid oil component-removing method, cell-separating method and cell-separating kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell-separating method by which cells can stably be separated from a body fluid such as marrow fluid, peripheral blood, cord blood, or menstrual blood, and to provide a cell-separating kit.SOLUTION: The liquid oil component-removing method is a method for removing a liquid oil component from a body fluid containing the component. When the liquid oil component-removing method is used together with a cell-separating device, cells can stably and efficiently be separated from a liquid oil component-containing body fluid such as marrow fluid, peripheral blood, cord blood, or menstrual blood. The cell-separating kit capable of separating the cells is constructed.

Description

  The present invention relates to a method for removing a component from a body fluid containing a liquid oil component, a cell separation method using the removal method, and a cell separation kit used for the separation method.

  In recent years, it has become clear that bone marrow fluid and umbilical cord blood have adherent stem cells that can differentiate into various cells such as bone, cartilage, muscle, and fat. (Patent Document 1, Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3) A method for efficiently separating and amplifying the cells is extremely important from the viewpoint of the development of regenerative medicine.

  Adherent stem cells have been reported to occur in bone marrow at a very low frequency of about 1 in 10,000 to 1 million adults (Non-Patent Document 4), and the cell fraction is separated. -Various methods for collecting after concentration have been studied. For example, Pittenger et al. Have found that differentiation precursor cells into fat, cartilage, and bone cells are present in a fraction having a specific gravity of 1.073 using the Ficoll-pack fractionation method, which is a density gradient centrifugation method (non-native). Patent Document 4). However, the separation method using Ficoll requires the operation of washing the cells several times using a centrifuge to separate the separation liquid from the cells, and the operability is complicated. Variations in efficiency occur and it is difficult to perform stable cell separation.

  As a method of performing cell separation more stably, a separation method using a device using a nonwoven fabric is also disclosed (Patent Document 2). However, when using a device using a nonwoven fabric, the cell separation efficiency is expected to greatly depend on the properties of the bone marrow fluid to be separated. For example, depending on the amount of the liquid oil component collected at the same time as the bone marrow fluid is collected, the surface of the nonwoven fabric is covered with the component and the original cell separation performance is not exhibited, or the liquid oil component adheres to the nonwoven fabric. Then, problems such as clogging and loss of target cells occur.

International Publication No. 01/83709 International Publication No. 07/046501

Pliard A. et al .: Conversion of an Immortilized Mesodermal Progenitor Cell Towards Osteogenic, or Adipogenic Pathways. J. Cell Biol. 130 (6): 1461-72 (1995) Mackay A. M. et al .: Chondrogenic differentiation of cultured human mesenchymal Stem Cell from Marrow.Tissue Engineering 4 (4): 415-428 (1998) Angele P. et al.:Engineering of Osteochondoral Tissue with Bone Marrow Mesenchymal Progenitor Cells in a Derivatived Hyaluronan Geration Composite Sponge.Tissue Engineering 5 (6): 545-553 (1999) Pittenger. Et al .: Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science 284: 143-147 (1999)

  An object of the present invention is to provide a cell separation method and cell capable of stably separating cells by removing the components from body fluids such as bone marrow fluid, peripheral blood, umbilical cord blood, and menstrual blood containing a liquid oil component It is to provide a separation kit.

  The present inventors diligently studied a method for stably separating cells from a body fluid containing a liquid oil component. As a result, the present inventors have found a method for removing a liquid oil component from a body fluid and combined it with a cell separation device to achieve the present invention that enables stable and efficient cell separation.

Therefore, the present invention is as follows.
(1) A method for separating cells from a body fluid, wherein the treatment is performed in the order of 1) liquid oil component removal step, 2) cell separation step,
(2) As a step of separating cells from body fluid, a cell separation device is prepared by filling a container having a liquid inflow portion and a liquid outflow portion with a cell separation material capable of capturing target cells, and the body fluid is passed through the device. The cell separation method according to (1), wherein the cells are separated by
(3) The liquid oil component removing step is characterized in that it is made to flow from the upper part of the chamber containing a liquid having a higher specific gravity than the component, and the body fluid from which the component has been removed is recovered from the lower part of the chamber (1 ) Or the cell separation method according to any one of (2),
(4) As a method for removing the liquid oil component, the body fluid containing the liquid oil component is placed in a container and allowed to stand, and the body fluid from which the component has been removed is recovered from the lower part of the container after oil-water separation. (1) or the cell separation method according to any one of (2),
(5) The cell separation method according to any one of (1) to (4), wherein the cell is a stem cell,
(6) A cell separation kit comprising at least a chamber for removing liquid oil components and a cell separation device, which are connected by a circuit,
(7) A cell separation method comprising using the cell separation kit according to (6),
(8) A body fluid containing a liquid oil component is introduced from the upper part of a chamber containing a liquid having a higher specific gravity than the component, and the body fluid from which the component has been removed is recovered from the lower part of the chamber. Oily oil component removal method,
(9) A liquid oil component removing method, wherein a body fluid containing a liquid oil component is placed in a container and allowed to stand, and the body fluid from which the component has been removed is recovered from the bottom of the container after oil-water separation,
(10) A biological fluid such as bone marrow fluid, umbilical cord blood, peripheral blood, menstrual blood, biological tissue such as adipose tissue liquefied by enzyme treatment, cells obtained from the liquefied material, physiological saline, cell culture medium, etc. The liquid oil according to any one of (8) and (9), comprising any one of a suspension resuspended in the buffer and a liquid component obtained simultaneously with adipose tissue during liposuction Component removal method.

  By using the liquid oil component removal method, cell separation method, and cell separation kit of the present invention, it becomes possible to stably and efficiently separate cells from a body fluid containing the liquid oil component.

It is a schematic diagram of a cell separation kit.

  Hereinafter, the present invention will be described in detail.

  The body fluid in the present invention refers to blood (including peripheral blood, G-CSF mobilized peripheral blood), bone marrow fluid, umbilical cord blood, menstrual blood, and the like, including cells. In addition, a living tissue such as adipose tissue liquefied by enzyme treatment or the like, and a cell obtained from the liquefied material resuspended in a buffer such as a physiological saline or a cell culture medium, during liposuction Liquid components obtained simultaneously with adipose tissue are also included.

  The liquid oil component in the present invention corresponds to an oil component that is substantially liquefied, and may have a paste form as long as it has fluidity, and may include an oil component on particles.

  The stem cells in the present invention refer to cells having pluripotency such as mesenchymal stem cells, pluripotent adult stem cells, bone marrow stromal cells, hematopoietic stem cells and the like.

  A mesenchymal stem cell refers to a cell that is isolated from a body fluid, has the ability to repeat self-proliferation, and can differentiate into a downstream cell lineage. A pluripotent stem cell refers to a cell that can be differentiated into a nerve cell or a hepatocyte by a differentiation-inducing factor, but is not limited thereto. Bone marrow stromal cells refer to all adherent cell components in bone marrow cells except immature and mature blood cells. A hematopoietic stem cell refers to a cell that can differentiate into a blood cell, for example, a white blood cell, a red blood cell, and a platelet. As mentioned above, although the stem cell in this invention was illustrated, this invention is not limited to this.

  The chamber in the present invention may be any container having a liquid inlet and a liquid outlet, and may have any form such as a sphere, a container, a cassette, a bag, a tube, and a column. Moreover, it is preferable that the liquid inflow port and the liquid outflow port are different from each other, and it is preferable that the liquid outflow port is disposed at the lowermost part when the chamber is installed in the vertical method. The chamber can be manufactured using any structural material.

  Specific examples of the structural material include non-reactive polymers, biocompatible metals, alloys, and glass. Non-reactive polymers include: acrylonitrile butadiene styrene terpolymer acrylonitrile polymers; polytetrafluoroethylene, polychlorotrifluoroethylene, copolymers of tetrafluoroethylene and hexafluoropropylene, halogenated polymers such as polyvinyl chloride; polyamides, Examples thereof include polyimide, polysulfone, polycarbonate, polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, polycarbonate acrylonitrile butadiene styrene, polystyrene, polymethylpentene, and the like.

  Examples of metal materials (biocompatible metals and alloys) include stainless steel, titanium, platinum, tantalum, gold, and alloys thereof, as well as gold-plated alloy iron, platinum-plated alloy iron, cobalt chromium alloy, and titanium nitride-coated stainless steel. Can be mentioned. As mentioned above, specific examples of the structural material of the chamber have been shown. Among them, a material having sterilization resistance is preferable. Specific examples include polypropylene, polyvinyl chloride, polyethylene, polyimide, polycardinate, polysulfone, and polymethylpentene. The chamber may be either soft or hard, and may be soft enough to be deformed by finger pressure.

  The liquid to be placed in the chamber of the present invention is not particularly limited as long as the specific gravity is heavier than that of the liquid oil component, but is preferably an isotonic liquid because cells in body fluid also pass through.

  For example, a physiological saline solution, Ringer's solution, a medium used for cell culture, a general buffer solution such as a phosphate buffer, and the like can be mentioned. From the viewpoint of safety, a physiological saline solution is preferable.

  In addition, the amount of liquid put into the chamber may be an amount that fills the chamber, or an air layer such as air may exist in the chamber. The speed of the body fluid flowing into the chamber is not particularly limited, but is preferably a speed at which the oil component floats on the upper portion of the chamber.

  Next, although the usage example of this chamber is described below, this invention is not limited to this. When body fluid containing liquid oil components flows into the chamber containing physiological saline and air from the liquid inlet located at the top of the chamber, the liquid oil components contain cellular components on the upper side of the isotonic solution. The body fluid is mixed with the isotonic solution and collected at the bottom of the chamber. The body fluid from which the liquid oil component has been removed flows out from the liquid outlet located at the bottom of the chamber and is collected. At this time, the target oil component is also contained in the liquid oil component, but the fluid interface is disturbed by the flow of body fluid into the chamber, and the cell component diffuses and collects in the lower part of the chamber. Therefore, efficient cell separation becomes possible.

  In the present invention, the container for standing the body fluid containing the liquid oil component may be any container that can hold the liquid, but preferably has a liquid inlet and a liquid outlet. The form may be any form such as a sphere, container, cassette, bag, tube, column, and the like. The container can be made using any structural material.

  Specific examples of the structural material include non-reactive polymers, biocompatible metals, alloys, and glass. Non-reactive polymers include: acrylonitrile butadiene styrene terpolymer acrylonitrile polymers; polytetrafluoroethylene, polychlorotrifluoroethylene, copolymers of tetrafluoroethylene and hexafluoropropylene, halogenated polymers such as polyvinyl chloride; polyamides, Examples thereof include polyimide, polysulfone, polycarbonate, polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, polycarbonate acrylonitrile butadiene styrene, polystyrene, and polymethylpentene.

  Examples of metal materials (biocompatible metals and alloys) include stainless steel, titanium, platinum, tantalum, gold, and alloys thereof, as well as gold-plated alloy iron, platinum-plated alloy iron, cobalt chromium alloy, and titanium nitride-coated stainless steel. Can be mentioned.

  Specific examples of the structural material of the container have been described above. Among them, a material having sterilization resistance is preferable. Specific examples include polypropylene, polyvinyl chloride, polyethylene, polyimide, polycardinate, polysulfone, and polymethylpentene.

  The size of the container is not particularly limited, but when the volume is excessively small, removal of the oil component is insufficient. Moreover, when the volume is excessively large, it leads to a loss of body fluid. From the above viewpoint, the volume of the container is preferably 0.5 to 100 mL, more preferably 3 to 50 mL, and still more preferably 5 to 30 mL.

  The standing time required for the oil / water separation in the present invention is only required to be able to substantially separate the oil / water, but there is a concern that the cell viability decreases when the treatment time is long. Accordingly, the standing time is preferably 1 to 60 minutes, more preferably 3 to 30 minutes, and more preferably 5 to 15 minutes.

  The cell separation material in the present invention can be separated from other cell components by capturing specific cell components. Examples of the material of the separating material include polyolefins such as polypropylene, polyethylene, high density polyethylene, and low density polyethylene, polyester, vinyl chloride, polyvinyl alcohol, vinylidene chloride, rayon, vinylon, polystyrene, acrylic (polymethyl methacrylate, polyhydroxyethyl methacrylate). , Polyacrylonitrile, polyacrylic acid, polyacrylate, etc.), nylon, polyurethane, polyimide, aramid, polyamide, cupra, kevlar, carbon, phenol, tetron, pulp, hemp, cellulose, kenaf, chitin, chitosan, glass, cotton, etc. What consists of at least 1 type chosen is preferable. More preferably, a synthetic polymer composed of at least one selected from polyester, polystyrene, acrylic, rayon, polyolefin, vinylon, nylon, polyurethane and the like can be mentioned.

  When two or more synthetic polymers are combined, the combination is not particularly limited, and preferred examples include polyester and polypropylene; rayon and polyolefin; or a combination of polyester, rayon and vinylon.

  As a form of the fiber when combining two or more kinds of synthetic polymers to form a fiber, a fiber in which one fiber is made of a synthetic polymer of different components, or a split fiber in which different components are separated from each other may be used. Moreover, the form which each compounded the fiber which consists of a synthetic polymer with a different component individually may be sufficient. The term “composite” as used herein is not particularly limited, and examples thereof include a form constituted by a state in which two or more kinds of fibers are mixed, or a form in which a form composed of a single synthetic polymer is bonded together. It is not limited to these.

  The form of the separating material is not particularly limited, and examples thereof include a porous body having a communication hole structure, an aggregate of fibers, and a woven fabric. Preferably it is comprised with a fiber, More preferably, it is a nonwoven fabric. The separating material preferably allows red blood cells to substantially pass therethrough. Here, the fact that red blood cells can substantially pass means that the passing rate of red blood cells with respect to the separating material is 80% or more.

  The cell separation device in the present invention is obtained by filling the cell separation material in a container having a liquid inlet and a liquid outlet. At this time, the cell separation material may be filled into a container without being compressed, or may be compressed into a container. The cell separating material is not limited in shape or the like as long as the conditions of the previous period are satisfied. There is no particular limitation on the form, size, and material of the container used for the cell separation device.

  The form of the container may be any form such as a sphere, a container, a cassette, a bag, a tube, or a column. Preferable specific examples include, for example, a cylindrical container having a capacity of about 0.1 to 400 ml and a diameter of about 0.1 to 15 cm; a square or rectangle having a length of about 0.1 to 20 cm and a thickness of 0.1. Examples include a square columnar container of about 5 cm, but the present invention is not limited to these.

  The container used for the cell separation device can be produced using any structural material. Specific examples of the structural material include non-reactive polymers, biocompatible metals, alloys, and glass.

  Non-reactive polymers include: acrylonitrile butadiene styrene terpolymer acrylonitrile polymers; polytetrafluoroethylene, polychlorotrifluoroethylene, copolymers of tetrafluoroethylene and hexafluoropropylene, halogenated polymers such as polyvinyl chloride; polyamides, Examples thereof include polyimide, polysulfone, polycarbonate, polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, polycarbonate acrylonitrile butadiene styrene, polystyrene, polymethylpentene, and the like.

  Examples of metal materials (biocompatible metals and alloys) include stainless steel, titanium, platinum, tantalum, gold, and alloys thereof, as well as gold-plated alloy iron, platinum-plated alloy iron, cobalt chromium alloy, and titanium nitride-coated stainless steel. Can be mentioned. As mentioned above, specific examples of the structural material of the container used in the device have been shown. Among them, a material having sterilization resistance is preferable. Specific examples include polypropylene, polyvinyl chloride, polyethylene, polyimide, polycardinate, polysulfone, and polymethylpentene.

  Next, although the usage example of this device is described below, this invention is not limited to this. First, by passing a body fluid from the liquid inlet of the cell separation device, red blood cells can flow out of the liquid outlet without being substantially captured, and target cells can be captured in the cell separation material. Next, it is possible to wash and remove the majority of red blood cells accumulated in the cell separation material by passing the washing solution from the same direction. Furthermore, the target cells can be separated and recovered with high efficiency by flowing the cell recovery liquid from the liquid outlet, that is, in the direction opposite to the direction in which the body fluid and the washing liquid are flowed.

  The cell separation kit in the present invention refers to one having the chamber and the cell separation device, which are connected by a circuit. In addition, you may have a bag that stores body fluids, a bag that stores cleaning fluid, a bag that stores fluid that flows out of the device, and a bag that collects recovered cells, which are closed by a circuit. Preferably it is.

  As the most preferable mode, referring to FIG. 1, the body fluid bag 3 and the washing solution bag 4 are arranged upstream of the chamber 1, and the cell separation device 2, the effluent bag 5 and the recovery bag 6 are arranged downstream of the chamber. Although the cell separation method using this kit is illustrated below, this invention is not limited to this.

1) Liquid oil component removal step;
First, the cleaning liquid is caused to flow from the cleaning liquid bag 4 so as to satisfy 50% of the chamber 1. At this time, the cleaning liquid may be naturally dropped from the cleaning liquid bag 4 through the circuits 12 and 13 or may be passed through a pump. In the case of liquid passing by an infusion pump, a probe for detecting droplets may be attached to the chamber 1.

  Next, the inside of the cell separation device 2 is also filled with physiological saline from the liquid outlet of the chamber 1. Next, the three-way cock 8 is switched to allow the body fluid to flow into the chamber 1 from the body fluid bag 3. At this time, the body fluid flows into the chamber 1 as droplets from the chamber 1 liquid inlet, but the liquid oil component is collected at the upper portion of the chamber 1 because of its low specific gravity.

  The target oil is also contained in the liquid oil component collected in the upper part of the chamber 1, but the liquid oil component collected by the body fluid dripped from the liquid inlet sequentially diffuses, so that the target cell diffuses into the body fluid without loss. It flows out from one liquid outlet.

2) cell separation step;
First, the body fluid that has flowed out of the chamber 1 flows into the cell separation device 2 through the circuits 14 and 16, and target cells are captured by the cell separation device 2.

  Next, a washing solution is passed through the chamber 1 and the cell separation device 2. The cleaning liquid may be sent naturally through the circuits 12, 13, 14, and 16, or may be passed through a pump. The amount of washing varies depending on the cell separation device volume, but washing is preferably performed at a volume of about 1 to 100 times the device volume.

  Examples of the cell washing solution include physiological saline, Ringer's solution, medium used for cell culture, and general buffers such as phosphate buffer, and physiological saline is preferable from the viewpoint of safety.

  Next, the cell collection liquid is put into the cell separation device 2 from the direction opposite to the direction in which the body fluid and the washing liquid are flowed (liquid outflow side), and the captured cells are collected.

  When the target cells are collected, the cell collection solution can be put in a syringe or the like in advance, and the plunger of the syringe can be pushed out by hand or the like. In this case, the cell recovery solution is caused to flow by connecting a syringe or the like to the recovery port 7. Although the amount of the cell recovery solution and the flow rate vary depending on the device volume, it is preferable to inject the cell recovery solution of about 1 to 100 times the device volume at a flow rate of about 0.5 to 20 ml / sec. It is not something.

  The cell recovery solution is not particularly limited as long as it is an isotonic solution, and examples thereof include those that have been used as injections such as physiological saline and Ringer's solution, buffer solutions, and cell culture media.

  Further, in order to increase the recovery rate of the cells captured by the cell separation device, the viscosity of the cell recovery solution may be increased. For that purpose, albumin, fibrinogen, globulin, dextran, hydroxyethyl starch, hydroxyethylcellulose, collagen, hyaluronic acid, gelatin and the like can be added to the cell recovery solution.

Hereinafter, as examples of the present invention, mesenchymal stem cell separation from bone marrow fluid will be described in detail, but the present invention is not limited to these examples.
[Example 1]
(1) Preparation of body fluid containing liquid oil component Anesthesia was performed by injecting ketalal and cerectal by intramuscular injection into domestic pigs weighing approximately 30 kg, and then adding nembutal by intravenous injection. Heparin was put in advance in a 10 ml syringe so as to be about 20 IU / ml, and bone marrow fluid was collected from the ilium using a 15 G bone marrow puncture needle.

  Next, heparin was additionally added to the collected bone marrow fluid pool to a final concentration of 50 IU / ml, and the mixture was thoroughly inverted. Next, bovine bone marrow fat (Taiko Co., Ltd.) and salad oil (Nisshin) were mixed at a volume ratio of 1: 1 as a liquid oil component. The liquid oil component was added so that the bone marrow fluid: liquid oil component = 6: 1, and the mixture was thoroughly inverted and mixed.

(2) Preparation of cell separation kit Non-woven fabric made of rayon and polyolefin as a cell separation material (weight per unit = 95 (g / m) on a cylindrical polycarbonate cylinder having an inner diameter of 2.2 cm and a length of 0.9 cm provided with an inlet / outlet ² ), fiber diameter = 15 ± 9 μ) was punched out into a circular shape having a diameter of 2.2 cm, 36 sheets were compressed and laminated, and the upper and lower sides were sandwiched between stoppers to prepare a cell separation device.

  Next, a liquid inlet was provided at the upper end of a cylindrical container made of vinyl chloride having a length of 6 cm and a diameter of 2 cm, and a liquid outlet was provided at the lower end to form a chamber. The chamber liquid outlet and the upper end of the cell separation device were connected by a circuit to obtain a cell separation kit. Moreover, the circuit at the upper end of the cell separation device was branched and a pressure gauge was installed.

(3) Performance evaluation After a physiological saline bag is connected to the liquid inlet of the chamber, 50% of the chamber volume is filled with physiological saline, and the inside of the cell separation device is also physiologically supplied from the liquid outlet of the chamber. Filled with saline solution. After removing the physiological saline bag, 35 ml of bone marrow fluid containing the liquid oil component prepared in (1) was passed at a flow rate of 6 ml / min. Next, 37 ml of physiological saline was flowed from the same direction at the same flow rate to wash away erythrocytes and the like. Between the bone marrow fluid passage and the washing fluid passage, the device inlet side pressure was measured with a pressure gauge as an index of clogging of the device.

  As a result, the device inlet side maximum pressure was 106 mmHg. Next, 100 ml of a cell culture solution (GIBCO α-MEM medium) containing 10% fetal calf serum was vigorously flowed in the direction opposite to the direction in which the bone marrow fluid was flowed, thereby collecting the captured cell fraction.

Next, a cell culture solution (GIBCO α-MEM medium) containing fetal bovine serum 10% is added to 1/30 volume of the collected cell suspension (equivalent to 1 ml of bone marrow fluid treatment) and transferred to a polystyrene dish (diameter 10 cm). The culture was performed at 37 ° C. in a 5% CO ₂ environment. After 9 days of culture, the colonies were stained with crystal violet-methanol solution, the number of colonies that appeared was measured, and the number of recovered colonies was determined. As a result, the number of recovered colonies was 90.
[Comparative Example 1]
The cell separation device inlet pressure and the number of recovered colonies were measured in the same manner as in Example 1 except that the chamber was not provided in the cell separation kit. As a result, the maximum pressure on the cell separation device inlet side was 266 mmHg, and the number of recovered colonies was 62.
[Example 2]
(4) Preparation of body fluid containing liquid oil component Anesthesia was performed by injecting ketalal and cerectal by intramuscular injection into domestic pigs weighing approximately 30 kg, and then adding nembutal by intravenous injection. Heparin was put in advance in a 10 ml syringe so as to be about 20 IU / ml, and bone marrow fluid was collected from the ilium using a 15 G bone marrow puncture needle.

  Next, heparin was added to the collected bone marrow pool so as to have a final concentration of 50 IU / ml, and the mixture was thoroughly inverted. Next, bovine bone marrow fat (Taiko Co., Ltd.) and salad oil (Nisshin) were mixed at a volume ratio of 1: 1 as a liquid oil component. The liquid oil component was added so that the bone marrow fluid: liquid oil component = 20: 1, and the mixture was thoroughly inverted and mixed.

(5) Production of cell separation device A cylindrical polycarbonate tube having an inner diameter of 2.2 cm and a length of 0.9 cm provided with an inlet / outlet, and a nonwoven fabric made of rayon and polyolefin as a cell separation material (mesh = 95 (g / m ² ), fiber diameter = 15 ± 9 μ) was punched out into a circular shape having a diameter of 2.2 cm, 36 sheets were compressed and laminated, and the upper and lower sides were sandwiched between stoppers to prepare a cell separation device. A circuit was provided at the upper end of the cell separation device and branched, and a pressure gauge was installed.

(6) Performance evaluation The nonwoven fabric was washed with a physiological saline solution about 20 times the volume of the cell separation filter. Next, 31.5 ml of bone marrow fluid containing the liquid oil component prepared in (4) is placed in a 50 mL syringe and placed at the upper end of the separation device and left to stand vertically for 10 minutes, and then at a flow rate of 6 ml / min. The liquid was passed. At this time, liquid passing was finished when it was visually confirmed that only the oil component remained in the syringe.

Next, 37 ml of physiological saline was flowed from the same direction at the same flow rate. Between the bone marrow fluid passage and the washing fluid passage, the device inlet side pressure was measured with a pressure gauge as an index of clogging of the device. As a result, the device inlet side maximum pressure was 25 mmHg.
[Comparative Example 2]
The cell separation device inlet side pressure was measured in the same manner as in Example 2 except that the sample was not allowed to stand before passing through the bone marrow fluid. As a result, the cell separation device inlet side maximum pressure was 118 mmHg.

  From the above results, by carrying out the liquid oil component removal method of the present invention and the cell separation method using the removal method, an increase in the inlet pressure of the cell separation device is suppressed, and the separation device is clogged. It was shown that can be suppressed.

  As a result, it was shown that cell separation can be performed without being affected by the oil component even when the body fluid containing the liquid oil component is processed by the separation device.

1. Chamber 2. Cell separation device 3. Body fluid bag 4. Washing fluid bag 5. Effluent bag 6. Recovery bag 7. Recovery port 8, 9, 10. Three-way stopcock 11-17. Circuit

Claims (10)

A method for separating cells from a body fluid, wherein the treatment is performed in the order of 1) liquid oil component removal step and 2) cell separation step. As a step of separating cells from bodily fluids, a cell separation device is prepared by filling a container having a liquid inflow portion and a liquid outflow portion with a cell separation material capable of capturing target cells. The cell separation method according to claim 1, wherein the cells are separated. 3. The liquid oil component removing step is characterized in that the body fluid from which the component is removed from the lower portion of the chamber is collected by flowing from the upper portion of the chamber containing a liquid having a higher specific gravity than the component. The cell separation method according to any one of the above. 2. The liquid oil component removing step is characterized in that a body fluid containing a liquid oil component is placed in a container and allowed to stand, and the body fluid from which the component has been removed is recovered from the bottom of the container after oil-water separation. Or the cell separation method in any one of 2. The cell separation method according to claim 1, wherein the cell is a stem cell. A cell separation kit comprising at least a liquid oil component removing chamber and a cell separation device, which are connected by a circuit. A cell separation method using the cell separation kit according to claim 6. A liquid oil component characterized in that a body fluid containing a liquid oil component is introduced from the upper part of a chamber containing a liquid having a higher specific gravity than the component, and the body fluid from which the component has been removed is recovered from the lower part of the chamber. Removal method. A method for removing a liquid oil component, wherein a body fluid containing a liquid oil component is placed in a container and allowed to stand, and the body fluid from which the component has been removed is recovered from the bottom of the container after oil-water separation. Body fluid is bone marrow fluid, umbilical cord blood, peripheral blood, menstrual blood, biological tissue such as adipose tissue liquefied by enzyme treatment, etc., and cells obtained from the liquefied material are used in buffers such as physiological saline and culture medium for cell culture The liquid oil component removal method according to claim 8 or 9, comprising any one of a resuspended suspension and a liquid component obtained simultaneously with adipose tissue during liposuction.

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